Alternative techniques in fish toxicity testing Anders Goksøyr Department of Molecular Biology University of Bergen Biosense Laboratories AS
Welcome message from author
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Alternative techniques in fish toxicity testing
Anders Goksøyr
Department of Molecular Biology
University of Bergen
Biosense Laboratories AS
Biomarkers
Non-invasive methods
In vitro testing
Corophium v.
Sediment reworkerTurbot
Fish
ACUTE TOXICITY
Acartia tonsaSkeletonema c.
Green algae
Log Pow
Log partition in octanol
partition in waterPartition
octanol-water
OCTANOL
WATERBIOACCUMULATION
POTENTIAL
HPLC
or
BIODEGRADATIONBiodegradation seawater with natural
bacteria fauna
O2
O22CO
O2H
O2H
O2H
2CO
2CO
Day 28
CARBONDIOXIDE + WATER + S + P + O + N....
seawater with natural
bacteria fauna
O2
O2
O2
O2
O2
O2
O2
O2Day 0
CHEMICAL + OXYGEN
R-OH
R-OH
R-OH OECD 306 sea water test
“Ecotoxicological testing of chemicals”
Hazard and risk modelling - DREAM
OSPAR standard test
program
Corophium v.
Sediment reworkerTurbot
Fish
ACUTE TOXICITY
Acartia tonsaSkeletonema c.
Green algae
Log Pow
Log partition in octanol
partition in waterPartition
octanol-water
OCTANOL
WATERBIOACCUMULATION
POTENTIAL
HPLC
or
BIODEGRADATIONBiodegradation seawater with natural
bacteria fauna
O2
O22CO
O2H
O2H
O2H
2CO
2CO
Day 28
CARBONDIOXIDE + WATER + S + P + O + N....
seawater with natural
bacteria fauna
O2
O2
O2
O2
O2
O2
O2
O2Day 0
CHEMICAL + OXYGEN
R-OH
R-OH
R-OH OECD 306 sea water test
Risk assessment
DosageOSPAR dataOil/water /distributionLong term studiesPhysical dataSite informationSea currentsEtc.
DREAMDose-related Risk and Effect
Assessment Model
EIF(Environmental impact factor)
0
25
50
75
100
log [dose]
% d
ead
LC50
The relevance of LC50
Effects on a biological system
Exposure
Molecule/cellOrgan
Individual
Population
Society
Ecosystem
Time
Exposure, health status and biomarker responses
Biomarkers
• various definitions have been described
• e.g. “any biological response (...) at the individual level or below demonstrating a departure from the normal status”*
• used in ecotoxicology, human toxicology and human medicine
* Walker et al. 2001: Principles of Ecotoxicology
Biomarkers
Biomarkers of exposure
levels of compound/metabolites in the organism
e.g. PAH: bile-fluorescence
Biomarkers of effect/response
mRNA/enzyme/protein levels in target organs or body fluids
e.g. PCB/dioxin/PAH: CYP1A; Cd, Zn, Hg, Cu: MT; DNA-damage
Biomarkers for susceptibility
genetic polymorphisms, physiological condition
e.g. CYP2D6 in drug metabolism in humans
Biomarker Pollutant
Inhibition of ALAD Pb
Induction of MT Cd, Hg, Cu, Zn
Inhibition of AChE OPs, carbamates
Induction of CYP1A Dioxins, PCBs, PAHs
Porphyrin profiles Several OCs
Retinol profiles OCs
DNA and hemoglobin adducts Largely PAHs
Induction of Vtg Estrogenic chemicals
Other serum enzymes Metals, OCs, PAHs
Stress proteins Metals, OCs
Immune responses Metals, OCs, PAHs
Specificity of biomarkers
Biomarkers in environmental monitoring:Is area A influenced by effluents with endocrine disrupting effects?
A B C
[Vtg]
Concl.: Yes, area A is contaminated with
environmental estrogens
Vtg-ELISATake blood samples and
analyze in Vtg-ELISA
AB
C
Sample fish from different areas, or
have fish in cages for 2-4 weeks
Vitellogenin induction downstream sewage treatment works
0,0
0,1
0,2
0,3
0,4
upstream downstream downstream
1 km
downstream
2 km
Vit
ello
gen
in (
mg
/ml p
lasm
a)
Vitellogenin (mean ± SEM; mg/ml) in plasma from rainbow trout caged immediately upstream or
downstream and one and two km downstream from a sewage treatment works. From Parkkonen et al., 2000.
vite
lloge
nin
(m
g/m
l)
CYP1A responses in liver of caged tilapia, Volta River, Ghana
0
500
1000
1500
2000
2500
C S2 S3 S4
CYP1A protein
Gadagbui & Goksøyr (1996)
Effluent testing with salmon - water treatment plant at oil terminal
Vtg ELISAs for OECD fish species
Biosense develops
quantitative Vtg-analyses
based on robust, sensitive,
and specific
immunoassays
Rainbow trout
(Oncorhynchus mykiss)
Fathead minnow
(Pimephales promelas)
Carp
(Cyprinus carpio)
Medaka
(Oryzias latipes)
Zebrafish
(Danio rerio)
Environmental toxicogenomics is a new approach to environmental toxicology. Environmental toxicogenomics allows us to identify and characterize genomic signatures of environmental toxicants as gene
and protein expression profiles. A major application of gene expression profiling is to understand (human) genetic variability and
susceptibility to disease.
Concept Statement, National Centre for Toxicogenomics, NIEHS (USA)
Environmental toxicogenomics and toxicoproteomics
The proteome - the functional genome
The proteome is the full picture of proteins expressed by the genome of a cell under given conditions
Can give us new knowledge about molecular interactions, metabolic processes, and novel biomarkers
Full exploitation must be based on genome information, but also on basal biological knowledge (molecular, physiological, ecological)
Strategy:
Individual samples
Preparative 2-DE
on large gels
Sequencing using ESI and MALDI
Xenopus laevis
Cyprinus carpio
MCF-7/MVLN cells
In vitro and in vivo exposures
EASYRING = Environmental Agent Susceptibility
Assessment Utilising Existing and Novel Biomarkers As Rapid Non-Invasive Testing Methods.
Identification by database
searches
Screening on mini 2-DE
Prepare synthetic peptide antibodiesELISA assay and kit development
Preparative 2-DE and MALDI-TOF MS - carp plasma
150
kDa
250
100
75
50
37
25
pH 4,5 7
Male, 64 ng EE2/l
Serine proteinase inhibitor CP9
Vitellogenin
Warm-temperature-acclimation-related-65kDa protein
Transferrin variant A
Alpha-1-antitrypsin homolog precursor
Zgc: 56023
Fast myotomal muscle actin
Myosin heavy chain, fast skeletal muscle
112
47
50 49
106
105
65 64
6920
787787
74
76816979
48
86
80
104
Tolfsen, Grøsvik et al., in prep.
2-DE western of Vtg in mucus from carp
Vtg is present in mucus of EE2-exposed carp
64 ng EE2/L
pH 4 7
250
150
100
75
50
37
kDa
25
64 ng EE2/L
pH 4 7
Control
pH 4 7
64 ng EE2/L 250
150
100
75
50
37
kDa
25
pH 4 7
Eidem et al.
Development of dipstick for endocrine disruption monitoring - non-disruptive sampling of fish mucus
In vitro alternatives
Many aspects of toxicity can be studied in in vitro systems
Primary culture vs cell lines vs reporter cells (e.g. CALUX)
Screening, mechanistic studies
QSAR - in silico
Important to know the limitations of the systems!
Toxicity identification and evaluation (TIE) of effluents for EDC effects
Expose fish hepatocytes (in vitro) to water extracts
Analyze Vtg levels
Yes/No
Analyze fractions of water extracts for Vtg effects (in vitro)/
levels of suspected compunds
OK
Identify sources - adjust production processes &
effluent treatment processes
HPLC fractionation
In vitro limitations
In vitro assays can rank chemicals within a given test, but they cannot be extrapolated or predict whether the test chemicals will maintain the same order of potency in a live animal bioassayIn vitro assessments for estrogenicity, particularly for chemicals which require metabolic activation or are capable of substantial
bioaccumulation, underestimate the responses observed with in vivo testingThe in vitro assays do not identify proestrogens, are insensitive to antiestrogens and, although they provide information on binding affinity to the ER, they do not provide any information regarding potential physiological alterationsIn vitro assays working at the biochemical and cellular level do not fully incorporate the signal amplification process observed in the exposure of whole organisms.
Folmar et al. (2002) A comparison of the estrogenic potencies of estradiol,
ethynylestradiol, diethylstilbestrol, nonylphenol and methoxychlor in vivo and in vitro.
Aquat. Toxicol. 60:101-110:
The brain-pituitary-gonad-
liver axis is a target for endocrine
disruptors
Arukwe & Goksøyr (2003) Comp Hepatol
Changes in embryonic sex ratios in eelpout caught near a pulp mill
Embryonic sex ratios in viviparous eelpout from four reference sites and four sites near a pulp mill outfall. The study comprises 3,423 embryos from 99 females.
From Larsson et al. (2000), Environ. Toxicol. Chem.
35
40
45
50
55
Kat
tega
t
Ska
gerrak
100
km n
orth
46 km
nor
th
1.7
km n
orth
1.2
km sou
th
4 km
sou
th
26 km
sou
th
% f
em
ale
em
bry
os
Effects of EE2 on germ cells and gonad differentiation in vasa-GFP transgenic medaka
Hano et al., ETC 24:70-77 (2005)
EE2 injection into
embryos caused
abnormal gonadal
development in both
sexes.
Complete sex reversal
in some XY males after
0.5-, 2.5-, and 5.0-ng
treatments.
No changes in XX
females after any
treatment.
Long-term effects
• Critical windows
• Accumulated effects
• Effects on fecundity and recruitment
• Transfer to and effects in offspring
• Population effects
The emergence of systems biology
The emerging field of systems biology attempts to harness the power of mathematics,
engineering, and computer science to analyze and integrate data from all the “omics” and
ultimately create working models of entire biological systems.
Spivey A.(2004) Systems biology: the big picture. Environ Health Perspect.
The three R’s
Replacement - to replace live animal studies with alternative methods, when possible
Reduction - to reduce the number of animals used in the study
Refinement - develop methods to ensure better animal welfare, reduced stress and improved quality of data obtained
Exit LC50 in fish toxicity testing?
In vitro testing, when possible- replacement
Non-destructive biomarkers - reduction
Sublethal toxicity tests with biomarker endpoints - refinement
• Bjørn Einar Grøsvik
• Christina Tolfsen
• Anneli Bohne Kjersem
• Lilian Sundbäck
• Tina Søfteland
• Biosense:
• Bente M. Nilsen
• Sven Inge Kristiansen
• Janne K. Eidem
• Institute of Marine Research
• Akvamiljø/RF
Support:
• Norwegian Research Council/Total
• EU (FP5): EASYRING project
Acknowledgements
EASYRING partners:Alberta Mandich, University of GenovaLuigi Viganó, IRSA-CNR, Milano, ItalyEmilio Benfenati, Mario Negri Institute, Milano, ItalyWerner Kloas, IGB-BerlinAnne van Cauwenberge, UMH, Mons, BelgiumMark Cronin, LJMU, Liverpool, UK
Related Documents
